Sulggi Lee PROJECT SUMMARY/ ABSTRACT The most commonly used illicit stimulant in HIV-infected individuals is methamphetamine (MA), and prior studies demonstrate strong evidence that MA promotes increased HIV transcription as well as immune dysregulation. HIV cure has emerged as an important clinical and research priority given evidence of ongoing immune dysfunction in HIV-infected individuals despite effective antiretroviral therapy (ART). A challenge in achieving worldwide HIV eradication is targeting specific vulnerable populations who are most likely to benefit from an HIV cure but possess poorer immune responses as a result of residual viral replication due to suboptimal ART adherence and/or direct immune dysfunction from illicit substance use. Prior non-human studies demonstrate that MA directly induces HIV production and promotes immune activation and inflammation. These preclinical findings suggest that HIV+ individuals who use MA may experience greater immune dysfunction and face additional challenges for future HIV eradication. The experiments of this R61/R33 proposal will investigate the effects of long-term and short-term MA exposure in HIV+ ART- suppressed individuals with a history of MA use. In Aim 1, HIV+ ART-suppressed participants with current MA use will be enrolled in a longitudinal cohort study to determine the effects of long-term MA exposure on residual virus production, gene expression, inflammation, and trace amine-associated 1 (TAAR1, a promising drug target for psychostimulant addiction) signaling. In Aim 2, HIV+ infected ART-suppressed individuals with a prior history of non-dependent MA use will be enrolled in an interventional study where they will be administered oral methamphetamine to determine the effects of short-term MA exposure on residual virus production, gene expression, inflammation, and TAAR1 signaling. MA exposure will be quantified in Aim 1 with hair samples, and in Aim 2 with multiple plasma samples collected over a 24-hour monitoring period. Measures of MA exposure will then be associated with residual virus production (HIV-1 cell-associated unspliced and multiply spliced RNA, and, as a marker of recent production ? episomal 2-LTR DNA), gene expression (validated by flow cytometry for protein expression), TAAR1 signaling (urinary ?-PEA levels), and inflammation (plasma interleukin-6, interleukin-10, tumor necrosis factor-?, and transforming growth factor-?). Using this approach, we will then functionally validate the top 60 genes identified in Aims 1 and 2 using a powerful gene editing technique (CRISPR-Cas9) using primary HIV+ CD4+ T cells. The proposed study will be the first human genetic study to directly evaluate the effect of MA exposure on residual viral transcription during effective ART. The overall goals of the study are to integrate a rigorous clinical study designs with high throughput `omics data to identify novel targets for reversing HIV latency, reducing inflammation, and personalizing future therapeutic strategies specific to HIV+ ART-suppressed individuals who use MA.